Non-enzymatic, Serum-free Tissue Culture of Pre-invasive Breast Lesions for Spontaneous Generation of Mammospheres

The overall goal of the following experiment is to culture Non enzymatically disrupted sterile breast tissue containing areas of insi ductal carcinoma or DCIS lesions to engender the spontaneous formation of mammo spheres in vitro. This is achieved by partially submerging small pieces of breast tissue containing ductal segments in a minimal volume of serum free medium to provide an oxygen and nutrient gradient within the organoid. Next, the organoids are cultured without disturbance until the epithelial cells and fibroblasts emerge from the open ducts and attach to the tissue culture flask.

Ultimately, the epithelial origin of the resulting spontaneous mam ophere formation can be confirmed by both direct microscopic examination and immunofluorescent staining. The main advantage of this technique over existing methods, which use enzymatic dissociation of the breast tissue, is that our breast DCIS organoid culture maintains the biological context of the ductal microenvironment without disrupting the in vivo cellular interactions. Although this method can provide unique insights into the biology of breast ductal carcinoma in situ two can also be applied to other types of preinvasive neoplastic lesions, such as pancreatic intraepithelial or prostatic intraepithelial neoplasia.

When performing this procedure, it is important to take care that the tissue remains sterile during the transport between the surgical suite and the tissue processing area. Furthermore, the instruments and reagents use for the tissue processing should be sterile to prevent contamination When grossing the tissue Before acquiring the tissue specimens first disinfect the work area with 70%ethanol and open a pair of sterile gloves, placing the wrapper on the work surface. Place the sterile interior of the glove wrapper, face up, and then put on the gloves.

Next, clean the surface of the specimen container with 70%ethanol and remove the plastic wrap from the container. Place the breast tissue specimen on the glove wrapper and then dip two cotton tip swabs into the tissue marking dye. Roll the swabs across the tissue surface to apply the dye to the tissue, blot the dyed tissue with a piece of vinegar soaked gauze, and then cut the breast tissue into roughly five millimeter thick vertical pieces without cutting all the way through the tissue.

Keeping the tissue sterile is the most important part of the procedure. To ensure success, it's important to have the dyes cotton tip swabs, sterile gauze vinegar and blades, arrange for easy access to avoid contamination during the tissue processing. Now palpate the tissue for areas of calcification, identifying the DCIS by their characteristic firm pale appearance surrounded by reddish rubbery borders that feel gritty due to calcium spicules.

Cut out any areas of calcification, including a small amount of surrounding breast tissue. Then place the tissue into a sterile 50 milliliter tube containing 20 to 30 milliliters of nutrient-rich medium. After inverting the tube several times to mix the tissue and medium, replace the supernatant with fresh medium.

Then place the tube into an insulated container and transport the tissue to the tissue culture lab to culture the breast tissue cells. Pour the tissue and a small amount of the nutrient rich medium into a sterile petri dish. Then use a sterile scalpel to cut away the fibrous tissue.

After discarding the fibrous pieces, cut the breast tissue into about three square millimeter pieces with each organoid containing at least one discernible duct segment within the surrounding stroma. Next, use sterile forceps to place the organoids into a sterile tissue culture flask and add 11 milliliters of freshly prepared serum free nutrient rich medium. Swirl the flask to evenly distribute the organoids throughout the medium, and then incubate the flask at 37 degrees Celsius and 5%CO2 for 48 hours.

On day two, post incubation, gently place the flask on an inverted microscope stage to check for potential bacterial or fungal contamination, taking care to avoid sudden sharp movements or swirling of the flask. If no contamination is noted, return the flask to the incubator for an additional day. If contamination is noted, discard the flask and contents in an appropriate container on day three, post incubation.

Place milliliters of medium in a sterile container at 37 degrees Celsius for 20 to 30 minutes. Then without disturbing the organoids, replace the conditioned medium with the prewarm fresh medium. Very gently rotate the flask to distribute the medium across the flask surface, and then place the flask back into the incubator.

Once the organoid cell colonies have been established, replace the medium every three days as just demonstrated. After 10 to 14 days in culture, discard any non-adherent pieces of tissue, mammo spheres and 3D structures arise spontaneously from multiple independent human DCIS duct tissue fragments from different patients diagnosed with atypical ductal hyperplasia or ductal carcinoma in situ, neither serum basement membrane extract nor gel-like matrices are required for spontaneous mammos sphere formation. The mammos spheres also generate mammary xenograft tumors in an NOD skid mouse model with the same growth pattern as that of invasive cancer.

These results demonstrate that progenitor cells with invasive potential preexist within the human breast, DCIS duct, but are apparently held in, checked by the ductal niche and can be coaxed to emerge in organoid culture. The epithelial origins of the mammos spheres and xenografts derived from DCIS mammo spheres can be confirmed by immunofluorescence. For example, these images demonstrate the identification of mammos spheres in an envivo culture by a mouse monoclonal antibody reactive to human epca, as well as at the center of an NOD skid xenograft.

The mammos sphere forming neoplastic epithelial cells in this representative culture were derived from preinvasive breast lesions that were devoid of Frank invasion or Microinvasion Histopathologic examination of the tissue used for organoid culture revealed confluent intraductal lesions with intact basement membrane boundaries, suggesting that the spontaneous mammo spheres formed in this culture system were derived only from preinvasive neoplastic areas, and were not a product of rare areas of microinvasion. While attempting this procedure, it's important to remember to communicate with radiologists, pathologists, and the research staff regarding the need to maintain the tissue sterility during the tissue grossing or imaging studies. Following this procedure, other methods like reverse phase protein microarrays or immunohistochemistry can be performed to answer additional questions.

For example, we can ask what is the molecular profile of the various cell populations that spontaneously emerge within this culture system? Our technique has paved the way for researchers in the field of proteomics and genomics to explore autophagy calcium signaling and DNA repair in living breast pre-malignant tissue.